Arginine Deprivation, Autophagy, Apoptosis (AAA) for the Treatment of Melanoma

The majority of melanoma cells do not express argininosuccinate synthetase (ASS), and hence cannot synthesize arginine from citrulline. Their growth and proliferation depend on exogenous supply of arginine. Arginine degradation using arginine deiminase (ADI) leads to growth inhibition and eventually cell death while normal cells which express ASS can survive. This notion has been translated into clinical trial. Pegylated ADI (ADI-PEG20) has shown antitumor activity in melanoma. However, the sensitivity to ADI is different among ASS(−) melanoma cells. We have investigated and reviewed the signaling pathways which are affected by arginine deprivation and their consequences which lead to cell death. We have found that arginine deprivation inhibits mTOR signaling but leads to activation of MEK and ERK with no changes in BRAF. These changes most likely lead to autophagy, a possible mechanism to survive by recycling intracellular arginine. However apoptosis does occur which can be both caspase-dependent or independent. In order to increase the therapeutic efficacy of this form of treatment, one should consider adding other agent(s) which can drive the cells toward apoptosis or inhibit the autophagic process

Arginine Deprivation as a Targeted Therapy for Cancer

Certain cancers may be auxotrophic for a particular amino acid and amino acid deprivation is one method to treat these tumors. Arginine deprivation is a novel approach to target tumors which lack argininosuccinate synthetase (ASS) expression. ASS is a key enzyme which converts citrulline to arginine. Tumors which usually do not express ASS include melanoma, hepatocellular carcinoma, some mesotheliomas and some renal cell cancers. Arginine can be degraded by several enzymes including arginine deiminase (ADI). Although ADI is a microbial enzyme from mycoplasma, it has high affinity to arginine and catalyzes arginine to citrulline and ammonia. Citrulline can be recycled back to arginine in normal cells which express ASS, whereas ASS(−) tumor cells cannot. A pegylated form of ADI (ADI-PEG20) has been formulated and has shown in vitro and in vivo activity against melanoma and hepatocellular carcinoma. ADI-PEG20 induces apoptosis in melanoma cell lines. However, arginine deprivation can also induce ASS expression in certain melanoma cell lines which can lead to in-vitro drug resistance. Phase I and II clinical trials with ADI-PEG20 have been conducted in patients with melanoma and hepatocellular carcinoma and antitumor activity has been demonstrated in both cancers. This article reviews our laboratory and clinical experience as well as others with ADI-PEG20 as an antineoplastic agent. Future direction in utilizing this agent is also discussed

Abstract LB-222: Identifying cFLIP as a marker and also a potentially “druggable” target of SAHA+TRAIL (TNF-Related Apoptosis Inducing Ligand), cytotoxicity in malignant pleural mesothelioma (MPM)

Abstract Background: Despite expressing adequate levels of receptors for TRAIL significant percentages of cancer cells are resistant to TRAIL-induced apoptosis. We have previously reported that histone deacetylase inhibitor SAHA (vorinostat) + TRAIL combination induces profound supra-additive cytotoxicity in MPM in vitro. We observe that only MPM cells with some TRAIL sensitivity are very susceptible to SAHA+TRAIL cytotoxicity. We hypothesize that TRAIL-initiated death signal at the membrane level determine cellular susceptibility to SAHA+TRAIL. The objective of this study is to identify the molecular marker that predicts TRAIL and thus TRAIL+SAHA sensitivity in MPM cells. Materials and methods: Intrinsic sensitivity of 8 MPM and primary normal endothelial cells to TRAIL and SAHA+TRAIL is determined by cell viability assay; basal expression of cFLIP, caspase 8, DR4/DR5, FADD, RIP, TRADD in these cells are evaluated by western blots. Selective knockdown of FLIP is achieved by siRNA. FLIP mRNA levels were determined by quantitative RT-PCR. Results: 5/8 MPM cells are very suceptible to SAHA+TRAIL cytotoxicity (combination-sensitive cells) while 3 others are classified as combination-resistant. Western blot analysis identifies an inverse relationship between cFLIP as well as procaspase 8 expression and sensitivity to SAHA+TRAIL with only the difference in cFLIP levels as quantified by densitometric analysis being distinctive between two groups: 0.87±0.02 for 5 combination-sensitive MPM cells versus 0.15±0.05 for 3 combination-resistant MPM cells. siRNA-mediated partial cFLIP knockdown restores DISC activity in high cFLIP expressing cells as evidenced by caspase 8 and 3 catalytic processing with stronger caspase activation being noted in combination-treated cells. Quantitative RT-PCR demonstrates high level of cFLIP messenger RNA in high cFLIP expressors indicating that cFLIP is transcriptionally upregulated in these cells. Selective cFLIP downregulation in high cFLIP expressing cells restores susceptibility to TRAIL and strongly sensitizes them to SAHA+TRAIL cytotoxicity. Additionally, selective complete cFLIP knockdown abrogates the need for SAHA to achieve profound TRAIL-mediated cell death in MPM cells regardless of their intrinsic cFLIP epxression. Conclusdion: Our study identifies cFLIP expression as a marker of cellular sensitivity to SAHA+TRAIL in MPM in vitro. More importantly, gene knockdown experiments provide the proof of concept that cFLIP is a pontential “druggable” target and downregulation of which sensitizes resistant cancer cells to TRAIL and SAHA+TRAIL. Ongoing works aim to validate this observation in a larger panel of novel MPM cells and to define treatments strategies to downregulate cFLIP in tumor cells expressing high levels of this antiapoptotic protein. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2011-LB-222</jats:p

Growth Hormone-Releasing Hormone in Lung Physiology and Pulmonary Disease

Growth hormone-releasing hormone (GHRH) is secreted primarily from the hypothalamus, but other tissues, including the lungs, produce it locally. GHRH stimulates the release and secretion of growth hormone (GH) by the pituitary and regulates the production of GH and hepatic insulin-like growth factor-1 (IGF-1). Pituitary-type GHRH-receptors (GHRH-R) are expressed in human lungs, indicating that GHRH or GH could participate in lung development, growth, and repair. GHRH-R antagonists (i.e., synthetic peptides), which we have tested in various models, exert growth-inhibitory effects in lung cancer cells in vitro and in vivo in addition to having anti-inflammatory, anti-oxidative, and pro-apoptotic effects. One antagonist of the GHRH-R used in recent studies reviewed here, MIA-602, lessens both inflammation and fibrosis in a mouse model of bleomycin lung injury. GHRH and its peptide agonists regulate the proliferation of fibroblasts through the modulation of extracellular signal-regulated kinase (ERK) and Akt pathways. In addition to downregulating GH and IGF-1, GHRH-R antagonist MIA-602 inhibits signaling pathways relevant to inflammation, including p21-activated kinase 1-signal transducer and activator of transcription 3/nuclear factor-kappa B (PAK1-STAT3/NF-&kappa;B and ERK). MIA-602 induces fibroblast apoptosis in a dose-dependent manner, which is an effect that is likely important in antifibrotic actions. Taken together, the novel data reviewed here show that GHRH is an important peptide that participates in lung homeostasis, inflammation, wound healing, and cancer; and GHRH-R antagonists may have therapeutic potential in lung diseases